Determinants at the N- and C-termini of Gα12 required for activation of Rho-mediated signaling

Background: Heterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the α-subunits Gα₁₂ and Gα₁₃, stimulate the monomeric G protein RhoA through interaction with a distinct subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs). The structural features that mediate interaction between Gα₁₃ and RhoGEFs have been examined in crystallographic studies of the purified complex, whereas a Gα₁₂:RhoGEF complex has not been reported. Several signaling responses and effector interactions appear unique to Gα₁₂ or Gα₁₃, despite their similarity in amino acid sequence.

Methods: To comprehensively examine Gα₁₂ for regions involved in RhoGEF interaction, we screened a panel of Gα₁₂ cassette substitution mutants for binding to leukemia-associated RhoGEF (LARG) and for activation of serum response element mediated transcription.

Results: We identified several cassette substitutions that disrupt Gα₁₂ binding to LARG and the related p115RhoGEF. These Gα₁₂ mutants also were impaired in activating serum response element mediated signaling, a Rho-dependent response. Most of these mutants matched corresponding regions of Gα13 reported to contact p115RhoGEF, but unexpectedly, several RhoGEF-uncoupling mutations were found within the N- and C-terminal regions of Gα₁₂. Trypsin protection assays revealed several mutants in these regions as retaining conformational activation. In addition, charge substitutions near the Gα₁₂ N-terminus selectively disrupted binding to LARG but not p115RhoGEF.

Conclusions: Several structural aspects of the Gα₁₂:RhoGEF interface differ from the reported Gα₁₃:RhoGEF complex, particularly determinants within the C-terminal α5 helix and structurally uncharacterized N-terminus of Gα₁₂. Furthermore, key residues at the Gα₁₂ N-terminus may confer selectivity for LARG as a downstream effector.